Use of Potato Virus X (PVX)–Based Vectors for Gene Expression and Virus‐Induced Gene Silencing (VIGS)

Christophe Lacomme1, Sean Chapman2

1 Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, United Kingdom, 2 Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, United Kingdom
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16I.1
DOI:  10.1002/9780471729259.mc16i01s8
Online Posting Date:  February, 2008
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Abstract

Potato virus X–based vectors are a well established system for rapid in planta studies. The vectors can be used for expression of proteins in plants and to down‐regulate genes through virus‐induced gene silencing. The development of binary‐based vectors for Agrobacterium delivery makes this system well suited to high‐throughput studies. Protocols are given for establishing infections to achieve expression and VIGS through in vitro transcription and Agrobacterium delivery to glasshouse and in vitro–grown plant material. Curr. Protoc. Microbiol. 8:16I.1.1‐16I.1.13. © 2008 by John Wiley & Sons, Inc.

Keywords: potato virus X; virus vector; gene silencing; Agrobacterium tumefasciens; solanaceous plants

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Infection of Plants with In Vitro Transcripts of Recombinant PVX
  • Basic Protocol 2: Infection of Plants by Agroinfiltration of Recombinant PVX Vector
  • Basic Protocol 3: Induction of VIGS in Potato Microtubers with Recombinant PVX Vectors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Infection of Plants with In Vitro Transcripts of Recombinant PVX

  Materials
  • pP2C2S‐derived recombinant plasmid (The Sainsbury Laboratory; http://www.ayeaye.tsl.ac.uk; follow links for David Baulcombe, then for “PVX vectors”)
  • SpeI restriction endonuclease and 10× buffer
  • 25:24:1 (v/v/v) phen4ol:chloroform:isoamyl alcohol saturated with 10 mM TE buffer, pH 8.0 ( appendix 2A)
  • 3 M sodium acetate pH 5.2 ( appendix 2A)
  • 100% and 70% ethanol
  • 10 mM TE buffer, pH 8.0 ( appendix 2A)
  • 0.8% and 0.7% nondenaturing agarose gels (Voytas, ) prepared in 1× TBE buffer ( appendix 2A)
  • 5× T7 RNA polymerase buffer
  • 0.1 M dithiothreitol (DTT)
  • 10× A/C/U/gTP mix (see recipe)
  • 5 mM cap analog (m7G[5′]ppp[5′]G)
  • 50 U/µl T7 RNA polymerase
  • 20 mM GTP
  • Plants to be infected (Table 16.1.1)
  • Aluminum oxide F400WP (Washington Mills Electro Minerals Company; http://www.washingtonmills.com/)
  • 0.5‐ml microcentrifuge tubes
  • Erlenmeyer flask
  • Gauze
  • Containment glasshouse or growth chamber
  • Additional reagents and equipment for agarose gel electrophoresis and ethidium bromide staining of gels (Voytas, )
    Table 6.0.1   MaterialsHost Plants Used for PVX‐Based Systemic Expression and Virus‐Induced Gene Silencing

    Genus Species Vectors Expression VIGS
    Nicotiana benthamiana P2C2S pGR106 + +
    clevelandii + Not tested
    paniculata + Not tested
    glutinosa + Not tested
    rustica + Not tested
    tabacum + Not tested
    Lycopersicon esculentum cultivars MoneyMaker, Marmande, Rossol, Rio Grande P2C2S pGR106 + Not tested
    pimpinellifolium + Not tested
    Solanum tuberosum cultivars Desiree, Bintje, Stirling P2C2S pGR106 + +
    bulbocastanum + +
    phureja accessions DB337, 71P10, 80CP23, 375/1 Not tested +
    phureja accessions DB271, DB371 Not tested
    chacoense accessions 3886, 5915, 7211, 7234 Not tested +
    Capsicum annuum P2C2S + Not tested

Basic Protocol 2: Infection of Plants by Agroinfiltration of Recombinant PVX Vector

  Materials
  • Electrocompetent A. tumefasciens strain GV3101 (Hellens et al., 2000) harboring pSoup plasmid (http://www.pgreen.ac.uk/JIT/JIT_fr.htm; follow links for “binary vectors,” then for “pGreen”)
  • pGR106 or pGR107‐derived recombinant plasmid
  • LB plates ( appendix 4A) with 50 µg/ml kanamycin
  • PVX‐ or insert‐specific primers (see information on supply and sequence data at http://www.ayeaye.tsl.ac.uk/; follow links for David Baulcombe, then for “PVX vector”; pGR106 and pGR107 GenBank accession numbers are, respectively, AY297843 and AY297842)
  • LB liquid medium ( appendix 4A) with 50 µg/ml kanamycin
  • Agromix (see recipe)
  • N. benthamiana plants (or other Solanaceae; see Table 16.1.1) to be inoculated
  • 28°C incubator
  • Bench‐top centrifuge
  • Spectrophotometer
  • 2‐ml syringes
  • Containment glasshouse or growth chamber
  • UV lamp: Blak‐Ray model B100AP (UVP) for green fluorescent protein visualization
  • Additional reagents and equipment for transformation of Agrobacterium (unit 16.2), the polymerase chain reaction (PCR; Kramer and Coen, ), and agarose gel electrophoresis (Voytas, )

Basic Protocol 3: Induction of VIGS in Potato Microtubers with Recombinant PVX Vectors

  Materials
  • pGR106 or pGR107‐derived recombinant plasmid in A. tumefasciens GV3101 harboring plasmid pSoup ( protocol 2)
  • In vitro–grown potato explants (Xu et al., ; in U.K. available from Scottish Agricultural Science Agency; http://www.sasa.gov.uk; ) propagated in potato explants propagation medium (see recipe) by internode cuttings (Xu et al., ; Hendriks et al., )
  • Microtuberization medium (see recipe)
  • Plant growth cabinet at 18° to 22°C with 16‐hr photoperiod and light intensity 110 µE m−2sec−1
  • Plant growth cabinet at 16°C with 8‐hr photoperiod and light intensity 80 µE m−2sec−1
  • Nescofilm (Karlan; http://www.karlan.com) or other laboratory film, e.g., Parafilm
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Figures

Videos

Literature Cited

   Baulcombe, D.C., Chapman, S., and Santa Cruz, S. 1995. Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 7:1045‐1053.
   Chapman, S., Kavanagh, T., and Baulcombe, D.C. 1992. Potato virus X as a vector for gene expression in plants. Plant J. 2:549‐557.
   Faivre‐Rampant, O., Gilroy, E.M., Hrubikova, K., Hein, I., Millam, S., Loake, G.J., Birch, P., Taylor, M., and Lacomme, C. 2004. Potato virus X‐induced gene silencing in leaves and tubers of potato. Plant Phys. 134:1308‐1316.
   Hellens, R., Mullineaux, P., and Klee, H. 2000a. Technical focus: A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5:446‐451.
   Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S., and Mullineaux, P.M. 2000b. pGREEN: A versatile and flexible binary Ti vector for Agrobacterium‐mediated plant transformation. Plant Mol. Biol. 42:819‐832.
   Hendriks, T., Vreugdenhil, D., and Stiekema, W.J. 1991. Patatin and four serine proteinase inhibitor genes are differentially expressed during tuber development. Plant Mol. Biol. 17:385‐394.
   Johansen, L.K. and Carrington, J.C. 2001. Silencing on the spot: Induction and suppression of RNA silencing in the Agrobacterium‐mediated transient expression system. Plant Phys. 126:930‐938.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
   Lu, R., Malcuit, I., Moffett, P., Ruiz, M.T., Peart, J., Wu, A.‐J., Rathjen, J.P., Bendahmane, A., Day., L., and Baulcombe, D.C. 2003. High throughput virus‐induced gene silencing implicates heat shock protein 90 in plant disease resistance. EMBO J. 22:5690‐5699.
   Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V., and Gleba, Y. 2004. In planta engineering of viral RNA replicons: Efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc. Natl. Acad. Sci. U.S.A. 101:6852‐6857.
   Ratcliff, F., Martin‐Hernandez, A.M., and Baulcombe, D.C. 2001. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25:237‐245.
   Takken, F.L.W., Luderer, R., Gabriels, S.H.E.J., Westerink, N., Lu, R., and deWit, P.J.G.M. 2000. A functional cloning strategy, based on a binary PVX expression vector, to isolate HR‐inducing cDNAs of plant pathogens. Plant J. 24:275‐283.
   Thomas, C.L., Jones, L., Baulcombe, D.C., and Maule, A.J. 2001. Size constraints for targeting post‐transcriptional gene silencing and for RNA‐directed methylation in Nicotiana benthamiana using a potato virus X vector. Plant J. 25:417‐425.
   Toth, R.L., Chapman, S., Carr, F., and Santa Cruz, S. 2001. A novel strategy for the expression of foreign genes from plant virus vectors. FEBS Letts. 489:215‐219.
   Santa Cruz, S., Chapman, S., Roberts, A.G., Roberts, I.M., Prior, D.A.M., and Oparka, K. 1996. Assembly and movement of a plant virus carrying a green fluorescent protein overcoat. Proc. Natl. Acad. Sci. U.S.A. 93:6286‐6290.
   Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D.C. 2003. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33:949‐956.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Xu, X., van Lammeren, A.A.M., Vermeer, E., and Vreugdenhil, D. 1998. The role of gibberellin, abscissic acid, and sucrose in the regulation of potato tuber formation in vitro. Plant Physiol. 117:575‐584.
Key Reference
  Chapman et al., . See above.
  Seminal paper describing construction and use of PVX‐based vectors for expression.
Internet Resources
  http://www.ayeaye.tsl.ac.uk
  Provides a source to obtain PVX‐based vectors. Also gives sequence information for vectors and methods for their use.
  http://www.pgreen.ac.uk/JIT/pSoup.htm
  Provide a source to obtain pSoup helper plasmid. Also gives sequence information for pSoup‐ and pGREEN‐derived vectors.
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